Mendelian statistics

I now have a single-insert, homozygous, transgenic plant line! This is a big deal because I can let plants of this line self-fertilize and all the progeny will also be transgenic, forever and ever. These plants will let me graduate!

I know because I counted the number of plants in each generation that grew in the presence of an antibiotic. In the first generation, there was just one plant, the parent of all the following generations. The seeds that this plant produced were mixed: 70% grew, and 30% died on the antibiotic. This was good news, because it meant only a single antibiotic resistance gene was inserted into the genome. Sometimes, with the methods I used, the resistance gene can be inserted more than once, which makes tracking the genotype much more difficult. If that had happened, more like 90% of the plants would have grown, and 10% would have died.

So of the 70% of plants that were resistant in the second generation, I saved 15, and let them self-fertilize to produce the third generation. Each of the 15 plants produced 10-40 seeds, and I sowed all of the seeds on media containing the antibiotic: about 500 seeds in all.

I counted, for each second-generation plant, how many seeds were resistant or not. Six of the 15 had all resistant seeds: they were homozygous. Nine of the fifteen had mixed seeds, with 72% resistant:  they were heterozygous. Now I am growing up plants from the homozygous bunch to get lots of seeds to continue experimenting with.

It is interesting to see how the statistics play out in real-life. According to Mendelian rules of inheritance the resistance of heterozygous lines should always be 75%, and probably, if I had used more like 200 seeds, I would have seen that percentage. But with only 40 seeds to sow, these numbers look pretty good. If I want to be most rigorous, I will have to apply the Chi-squared test.

It is also interesting because I have followed this procedure not with just one plant line (meaning a first-generation plant) but 12: 6,000 seeds! And this is for just one transgene - I have 4 so far that I am making: 24,000 seeds! Each seed is placed by hand (my hand!) on the media. And this number of 24,000 is not quite sufficient to determine if the plant line is single-insert and homozygous just from counting the number of resistant plants. If I had plenty of seeds, I should look at 5 times as many: 120,000 seeds!

Mendel was a very patient man, and I am glad I have the Chi-squared test to rely on.